Glial cells are crucial for the normal functioning of the brain, communicate extensively with neurons, and are affected by ethanol exposure, yet their role in the development of alcohol use disorders is poorly understood. The goal of this proposal is to study the contribution of glia to ethanol behavioral responses in the fruit fly Drosophila. A microarray study of gene regulation by ethanol exposure coupled with a genetic screen of flies carrying mutations in ethanol-responsive genes led to the identification of novel genes required for ethanol tolerance. Preliminary studies of one of these, the A-kinase anchoring protein Akap200, indicate that it is exclusively localized to glia in adult flies and that its function is required in glia to promote the development of ethanol tolerance. AKAP proteins spatially and temporally coordinate protein kinase A signaling with other cell signaling pathways, including protein kinase C, and we found that both protein kinase A and protein kinase C, like Akap200, can promote ethanol tolerance in glia. Additionally, we found that the glial blood-brain barrier, where Akap200 is prominently expressed, responds to ethanol exposure by changing its morphological and functional properties, and that barrier function is partially compromised in Akap200 mutants. We propose that glia regulate ethanol tolerance through conserved signaling pathways that are coordinated by Akap200. There are three main goals of our proposed research. First, we plan to characterize the response of each class of central nervous system glia to ethanol exposure by determining the time and dose-dependent effects of ethanol on glial structure/function, and the role played by glial cellular processes in the development of ethanol behavioral responses. Second, we plan to characterize the role of Akap200 and Akap200-interacting proteins in the development of ethanol tolerance and glial structure/function in order to establish the mechanisms by which glia regulate ethanol behavioral responses. Third, we plan to carry out a genetic screen for novel glial genes that regulate ethanol behavioral responses. The recent availability of tools for genetically targeting specific glial types, for limiting genetic manipulations to adults, and methodology for assessing glial structure and function, coupled with our discovery of a glial protein kinase A signaling pathway for ethanol tolerance, has given us an opportunity to make rapid progress in understanding the contributions of glia to ethanol behavioral responses. Evolutionary conservation of protein kinase A signaling and many glial properties suggests that what is learned in Drosophila can transfer to mammalian systems and may impact the treatment of alcohol use disorders in humans. PUBLIC HEALTH RELEVANCE: Glial cells are critically important for the normal functioning of the brain and they show complex and dynamic responses to ethanol, but their role in the development of alcohol use disorders remains to be characterized. We found that glia in the fruit fly Drosophila alter their shape and function in response to ethanol, and that evolutionarily conserved signal transduction pathways act in fly glia to regulate ethanol tolerance. Sophisticated genetic tools, a high level of molecular conservation, and similar glial properties make Drosophila an ideal system for uncovering molecular mechanisms that can provide novel insights in vertebrates, including humans.